CN106480198B - A kind of method and system carrying out individual identification to unknown sample - Google Patents
A kind of method and system carrying out individual identification to unknown sample Download PDFInfo
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- CN106480198B CN106480198B CN201610935444.3A CN201610935444A CN106480198B CN 106480198 B CN106480198 B CN 106480198B CN 201610935444 A CN201610935444 A CN 201610935444A CN 106480198 B CN106480198 B CN 106480198B
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The present invention provides a kind of method and system that individual identification is carried out to unknown sample, this method includes extracting unknown sample DNA, obtaining the DNA, the genotyping result of totally 31 locus, 30 InDel locus are rs10629077, rs2308026, rs361519, rs2307652, rs16671, rs33948716, rs8190507, rs1610937, rs2307689, rs2307976, rs1305056, rs2067353, rs1610963, rs2307632, rs178784 including 30 InDel locus and 1 sex identification locus Amelogenin 44, rs140847, rs2307554, rs2308020, rs1610902, rs2307839, rs2307708, rs2308115, rs3047269, rs2067294, rs2307553, rs16438, rs2307981, rs4646006, rs2308072 and rs140864；Individual identification is carried out according to the genotype of 31 locus of unknown sample.The solution of the present invention can realize the individual identification to sample unknown in Chinese population using InDel locus.
Description
Technical field
The present invention relates to a kind of method and systems of individual identification, more particularly to one kind to carry out individual identification to unknown sample
Method and system.
Background technique
Insertion-deletion polymorphism (insertion-deletion, InDel) is the insertion of section of DNA segment or is lacked
Lose two polymorphic alleles for being formed by specific type.There is following feature relative to STR and SNP, InDel: (1) wide
General is distributed in whole gene group；(2) because of single mutational events, and occurrence frequency is low, more stable after generation, uneasy to recur
Mutation；(3) first ancestor's informative site can be become, judge region source；(4) InDel can be expanded in lesser amplicon, be fitted
Detection for degradation of dna；(5) InDel can use the existing instrument and equipment of a forensic DNA laboratory and carry out Genotyping；(6)
InDel can be adapted for automation and high-throughput technology simultaneously.
Therefore, concern of the InDel by domestic and foreign scholars, and existing multidigit scholar establishes suitable for more to this nationality
The compound system of state property research.But due to various nationalities' allele multiplicity in Chinese population, how to establish a set of based on InDel
Locus, the identification of Forensic DNA suitable for extensive Chinese population composite amplification system makes substitution STR detection
Another powerful measure of system anlysis DNA sample, becomes problem to be solved.
Summary of the invention
The present invention provides a kind of methods for carrying out individual identification to unknown sample, and can obtain unknown sample includes 30
InDel locus and 1 sex identification locus genotyping result of totally 31 locus inside, to realize to unknown sample
Individual identification.
The present invention also provides a kind of systems for carrying out individual identification to unknown sample, may be implemented by the system to unknown
Sample is directed to the accurate parting of above-mentioned 31 locus, to carry out individual identification to unknown sample.
The present invention also provides a kind of compound detection system, it includes 30 that the detection architecture, which can accurately obtain unknown sample,
InDel locus and 1 sex identification locus genotyping result of totally 31 locus inside.
The present invention also provides a kind of detection kits, including the compound detection system.
A kind of method that individual identification is carried out to unknown sample provided by the invention, this method comprises:
1) DNA of unknown sample is extracted；
2) DNA is obtained totally 31 including 30 InDel locus and 1 sex identification locus Amelogenin
The genotyping result of a locus, 30 InDel locus be rs10629077, rs2308026, rs361519,
rs2307652、rs16671、rs33948716、rs8190507、rs1610937、rs2307689、rs2307976、
rs1305056、rs2067353、rs1610963、rs2307632、rs17878444、rs140847、rs2307554、
rs2308020、rs1610902、rs2307839、rs2307708、rs2308115、rs3047269、rs2067294、
Rs2307553, rs16438, rs2307981, rs4646006, rs2308072 and rs140864；
3) individual identification is carried out according to the genotype of 31 locus of unknown sample.
In the solution of the present invention, 31 locus are that applicant passes through the living environment to Chinese population, race
The progress such as origin comprehensive analysis, the phenotypic characteristic difference of investigation each department nationality population, including resemblance, physical signs etc.,
For these differences carry out document and network data base investigation, obtained on the basis of existing research can be carried out individual identification and
The combination of the specific gene seat of paternity identification.The unknown sample can for from human body blood sample, cast-off cells, bone,
The individual source of tooth, seminal stain and buccal swab equal samples, these samples is unknown.
It is described 2) including using one-to-one with 31 locus in the specific embodiment of the present invention
The step of 31 pairs of amplimers expand to obtain amplified production it；The amplimer is SEQ ID in sequence table
The nucleotide sequence of No.1 to SEQ ID No.62.
In another embodiment of the invention, wherein 2) further including using heredity after obtaining amplified production
The step of analyzer analyzes the amplified production, genotype to obtain 31 locus.It is described in the solution of the present invention
Genetic analyzer can be the conventional use of genetic analyzer of those skilled in the art, such as ABI3130 or ABI3500 type heredity
Analyzer passes throughID-X software or other GeneMapper softwares etc. analyze institute in the pcr amplification product
State the genotype of 31 locus.
A kind of system that individual identification is carried out to unknown sample provided by the invention, the system comprises DNA extraction system,
Compound detection system, and infer system；The DNA extraction system is used to extract the DNA of unknown sample；The compound detection body
System is for obtaining the DNA totally 31 bases including 30 InDel locus and 1 sex identification locus Amelogenin
Because of the genotyping result of seat, 30 InDel locus be rs10629077, rs2308026, rs361519, rs2307652,
rs16671、rs33948716、rs8190507、rs1610937、rs2307689、rs2307976、rs1305056、
rs2067353、rs1610963、rs2307632、rs17878444、rs140847、rs2307554、rs2308020、
rs1610902、rs2307839、rs2307708、rs2308115、rs3047269、rs2067294、rs2307553、
Rs16438, rs2307981, rs4646006, rs2308072 and rs140864；
The deduction system is used to carry out individual identification according to the genotype of 31 locus of unknown sample.
In the specific embodiment of the present invention, the compound detection system is used to use and 31 locus
One-to-one 31 pairs of amplimers expand it to obtain amplified production, and the amplimer is SEQ ID in sequence table
The nucleotide sequence of No.1 to SEQ ID No.62.
Further, the compound detection system is also used to after obtaining amplified production, is analyzed using genetic analyzer
The amplified production, to obtain the genotype of 31 locus.
A kind of compound detection system provided by the invention, the system include unknown sample DNA, 31 locus, and
Amplimer, the compound detection system include 30 InDel locus and 1 sex identification gene for obtaining the DNA
The genotyping result of seat Amelogenin totally 31 locus inside, 30 InDel locus be rs10629077,
rs2308026、rs361519、rs2307652、rs16671、rs33948716、rs8190507、rs1610937、
rs2307689、rs2307976、rs1305056、rs2067353、rs1610963、rs2307632、rs17878444、
rs140847、rs2307554、rs2308020、rs1610902、rs2307839、rs2307708、rs2308115、
Rs3047269, rs2067294, rs2307553, rs16438, rs2307981, rs4646006, rs2308072 and
rs140864；The amplimer by forming with the one-to-one 31 pairs of amplimers of 31 locus, draw by the amplification
Object is the nucleotide sequence of SEQ ID No.1 to SEQ ID No.62 in sequence table.
In the solution of the present invention, the archaeal dna polymerase can be Fast Start archaeal dna polymerase, Taq DNA polymerization
One of enzyme, Hotstart archaeal dna polymerase are a variety of.
The present invention also provides a kind of detection kits, including the compound detection system.
In the solution of the present invention, the present invention carries out the side of the parting of 31 locus using the compound detection system
Method, comprising: 1) using the unknown sample DNA of extraction as template；2) use the amplimer to the unknown sample as template
DNA carries out multiplexed PCR amplification reaction to obtain amplified production；3) amplified production is analyzed using genetic analyzer,
To obtain the genotyping result of 31 locus.
The present invention program has the advantage that
1, method and system of the invention carries out individual identification using specific 31 locus, these locus are in the Chinese
Race, Kazak, the Dai nationality, in the national genetic polymorphism sex investigation of Miao ethnic group and five, the Yao nationality, accumulating personal discrimination is respectively
0.999999999957,0.999999999990,0.999999999974,0.999999999875 and 0.999999999966,
Cumulative individual discrimination with higher.
2, method and system of the invention is suitable for extensive Chinese population, can degrade for forensic science laboratory testing
Sample provides effective technical support, while reducing testing cost, becomes substitution STR detection architecture and carries out DNA sample analysis
Another powerful measure.
3, scheme provided by the invention effectively can realize the deduction to unknown sample source from gene level, be Chinese population
The accurate scientific basis of offers such as individual identification and the identification of parental right relationship.
Detailed description of the invention
Fig. 1 utilizes the parting map of composite amplification system test stone DNA9947 sample.
Specific embodiment
The fresh peripheral venous samples of 421 parts of independent individuals used in the following embodiment, wherein 94 parts of Han nationality in Beijing, cloud
97 parts of southern the Dai nationality, 95 parts of Kazak ethnic population, 69 parts of Guangxi Miao, 66 parts of the Guangxi Yao nationality is mentioned by Material Evidence Identification Center, Ministry of Public Security
For.
DNTP (10mM) used in the following embodiment, 10 × PCR buffer (15mM Mg2+)、10×PCR buffer
(15mM Mg2+), quick start enzyme Hotstar Taq plus (5U/ μ l), human standard product (9947,1ng/ μ l), in molecular weight
Mark Typer500 is purchased from Material Evidence Identification Center, Ministry of Public Security, and POP7 running gel, deionized formamide are purchased from American AB company,
Fluorescent marker PCR primer is synthesized by the raw work in Shanghai.9700 type PCR instruments, 3130XL genetic analyzer are American AB company.
Embodiment 1, the verifying to the method and system accuracy of the invention for carrying out individual identification to unknown sample
In the present embodiment, the unknown sample is the fresh peripheral venous samples of 421 parts of independent individuals, wherein Bei Jinghan
94 parts of race, 97 parts of Yunnan the Dai nationality, 95 parts of Kazak ethnic population, 69 parts of Guangxi Miao, 66 parts of the Guangxi Yao nationality, it is known that its individual comes
Source, but it is unknown to set in the implementation process of the embodiment of the present application 1 its individual source, using the application method and system to its into
Row individual identification, comprising:
1) DNA of unknown sample is extracted using the DNA extraction system in system of the invention, 2) utilize system of the invention
In compound detection system to obtain the DNA include that 30 InDel locus and 1 sex identification locus Amelogenin exist
The inside genotyping result of totally 31 locus, 3) system is inferred using described in system of the invention, according to 31 bases of unknown sample
Because the genotype of seat carries out individual identification.
In the present embodiment, the compound detection system includes unknown sample DNA, 31 locus and amplimer, institute
Compound detection system is stated for obtaining the DNA totally 31 including 30 InDel locus and 1 sex identification locus
The genotyping result of locus, 30 InDel locus be rs10629077, rs2308026, rs361519,
rs2307652、rs16671、rs33948716、rs8190507、rs1610937、rs2307689、rs2307976、
rs1305056、rs2067353、rs1610963、rs2307632、rs17878444、rs140847、rs2307554、
rs2308020、rs1610902、rs2307839、rs2307708、rs2308115、rs3047269、rs2067294、
Rs2307553, rs16438, rs2307981, rs4646006, rs2308072 and rs140864；The amplimer by with institute
The one-to-one 31 pairs of amplimers composition of 31 locus is stated, the amplimer is SEQ ID No.1 to SEQ in sequence table
The nucleotide sequence of ID No.62.
1, the DNA of sample to be detected is extracted as template
According toDNA Blood Midi Kit specification (Qiagen, Germany) extracts blood sample DNA.It is all
After DNA is quantified through Nanodrop2000c (Thermo Scientific, the U.S.), 0.5ng/ μ l- is diluted to ultrapure water
1ng/ μ l is used.
2, the parting of 31 locus is carried out using the compound detection system, comprising: make the unknown sample DNA of extraction
For template；Multiplexed PCR amplification reaction is carried out using DNA profiling of the amplimer to extraction, to obtain amplified production；It will expand
Increase the genotyping result that product utilization genetic analyzer determines 31 locus.
Detailed process is as follows:
2.1, primer pond configures
The configuration in amplimer pond, wherein being the one-to-one 31 pairs of amplifications of 31 locus in the amplimer
Primer, in the present embodiment, it is preferred that the amplimer of 31 locus is SEQ ID No.1 to SEQ ID in sequence table
The nucleotide sequence of No.62；Various primer sequences provided by the invention are by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd
Synthesis.
Synthetic primer is diluted to 100 μM with 1 × TE buffer, by the upstream and downstream primer of 31 locus according to
Lower volume and concentration carry out mixing as 31 heavy PCR primer ponds (i.e. PrimerMix).
2.2, multi-PRC reaction
The present embodiment carries out multi-PRC reaction using 9700 type PCR amplification instruments.
(1) PCR mix (10 μ L system) is configured, as shown in table 5 below.
Table 5
(2) amplification program
The thermal circulation parameters of PCR amplification process are as follows: 95 DEG C of 11min；94 DEG C of 30s, 60 DEG C of 120s, 72 DEG C of 90s, totally 30 are followed
Ring；60 DEG C of extension 60min.
2.3, PCR product parting
1 μ L PCR product and 9.5 μ L deionized formamides, Typer500 internal standard are taken to mix, ice bath immediately after 95 DEG C of 3min
5min.Amplified production is passed through using ABI 3130XL type genetic analyzerID v3.2 software is divided
Analysis obtains the genotype of 31 locus.
2.4, interpretation of result
In order to verify the accuracy of genotyping result, 50 parts of DNA samples are randomly selected from 421 parts of DNA samples, to 31 bases
Because (sequencing of Beijing Mai Aodeen Biotechnology Co., Ltd) is sequenced in seat, obtained using the compound detection system of the present embodiment
All genotyping results it is consistent with sequencing result, consistency reaches 100%, this result is demonstrate,proved compound detection system of the present invention and divided
Type result is accurate.
It the use of standard DNA 9947 is simultaneously template using system progress PCR amplification of the invention, and to its 31 locus
Parting is carried out, genotyping result is as shown in Figure 1, consistent with DNA9947 sequencing result.
3, individual identification is carried out according to the genotype of 31 locus of unknown sample
By the individual source results for above-mentioned 421 parts of samples that the present embodiment method obtains, with its known to individual source tie
Fruit is consistent, illustrates that the method for the present invention can carry out individual identification to unknown sample.
Balance check of 30 InDel locus in different nationalities in the detection system of the present invention of embodiment 2
The genotyping result of the independent individuals of 421 people, and base are obtained according to 1 the method for embodiment using present system
In individual identification rate of 30 locus in various nationalities, (6) DP value is shown in Table, calculate 30 autosome InDel locus and exist
Han nationality, Kazak, the Dai nationality, the national cumulative individual discrimination (TDP) of Miao ethnic group and five, the Yao nationality.
Table 6
Cumulative individual discrimination calculation formula are as follows:
TDP=1- (1-DP1)(1-DP2)(1-DP3)…(1-DPK), wherein DPKFor the DP value of k-th of locus.
The detection system of the application substantially increases the cumulative individual discrimination to individual, in Han nationality, Kazak, the Dai Nationality
In race, the national genetic polymorphism sex investigation of Miao ethnic group and five, the Yao nationality, accumulate personal discrimination be respectively 0.999999999957,
0.999999999990,0.999999999974,0.999999999875 and 0.999999999966, heredity with higher is more
State property and cumulative individual discrimination.
Sequence table
<110>Material Evidence Identification Center, Ministry of Public Security
<120>a kind of method and system that individual identification is carried out to unknown sample
<130> 165944GF
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cagatcagca aaggctggta 20
<210> 56
<211> 20
<212> DNA
<213>artificial sequence
<220>
<223>primer
<400> 56
gtttggaaag aaaggcagga 20
<210> 57
<211> 20
<212> DNA
<213>artificial sequence
<220>
<223>primer
<400> 57
gcacccagcc ttctccttat 20
<210> 58
<211> 25
<212> DNA
<213>artificial sequence
<220>
<223>primer
<400> 58
gtggttcatt tcagactaca actca 25
<210> 59
<211> 20
<212> DNA
<213>artificial sequence
<220>
<223>primer
<400> 59
accacaggca aatcctgaag 20
<210> 60
<211> 20
<212> DNA
<213>artificial sequence
<220>
<223>primer
<400> 60
gggttaggga ggttggttga 20
<210> 61
<211> 19
<212> DNA
<213>artificial sequence
<220>
<223>primer
<400> 61
ccctgggctc tgtaaagaa 19
<210> 62
<211> 20
<212> DNA
<213>artificial sequence
<220>
<223>primer
<400> 62
gagcttaaac tgggaagctg 20
Claims (8)
1. the method that the unknown sample of a kind of pair of Chinese population carries out individual identification, which is characterized in that this method comprises:
1) DNA of the unknown sample is extracted；
2) DNA totally 31 bases including 30 InDel locus and 1 sex identification locus Amelogenin are obtained
Because of the genotyping result of seat, 30 InDel locus be rs10629077, rs2308026, rs361519, rs2307652,
rs16671、rs33948716、rs8190507、rs1610937、rs2307689、rs2307976、rs1305056、
rs2067353、rs1610963、rs2307632、rs17878444、rs140847、rs2307554、rs2308020、
rs1610902、rs2307839、rs2307708、rs2308115、rs3047269、rs2067294、rs2307553、
Rs16438, rs2307981, rs4646006, rs2308072 and rs140864；
3) individual identification is carried out according to the genotype of described 31 locus of unknown sample.
2. the method according to claim 1, wherein 2) described includes using and described 31 locus, one a pair
The step of 31 pairs of amplimers answered expand to obtain amplified production it；The amplimer is SEQ ID in sequence table
The nucleotide sequence of No.1 to SEQ ID No.62.
3. according to the method described in claim 2, it is characterized in that, wherein 2) further including using something lost after obtaining amplified production
It passes analyzer and analyzes the amplified production, the step of genotype to obtain 31 locus.
4. a kind of system for carrying out individual identification to unknown sample, which is characterized in that the system comprises DNA extraction systems, multiple
Detection architecture is closed, and infers system；
The DNA extraction system is used to extract the DNA of unknown sample；
The compound detection system includes 30 InDel locus and 1 sex identification locus for obtaining the DNA
The genotyping result of Amelogenin totally 31 locus inside, 30 InDel locus be rs10629077,
rs2308026、rs361519、rs2307652、rs16671、rs33948716、rs8190507、rs1610937、
rs2307689、rs2307976、rs1305056、rs2067353、rs1610963、rs2307632、rs17878444、
rs140847、rs2307554、rs2308020、rs1610902、rs2307839、rs2307708、rs2308115、
Rs3047269, rs2067294, rs2307553, rs16438, rs2307981, rs4646006, rs2308072 and
rs140864；
The deduction system is used to carry out individual identification according to the genotype of 31 locus of unknown sample.
5. system according to claim 4, which is characterized in that the compound detection system is used to use and 31 bases
Because the one-to-one 31 pairs of amplimers of seat expand it to obtain amplified production, the amplimer is in sequence table
The nucleotide sequence of SEQ ID No.1 to SEQ ID No.62.
6. system according to claim 5, which is characterized in that the compound detection system is also used to obtaining amplified production
Afterwards, the amplified production is analyzed using genetic analyzer, to obtain the genotype of 31 locus.
7. a kind of compound detection system, which is characterized in that the system includes unknown sample DNA and amplimer,
The compound detection system includes 30 InDel locus and 1 sex identification locus for obtaining the DNA
The genotyping result of Amelogenin totally 31 locus inside, 30 InDel locus be rs10629077,
rs2308026、rs361519、rs2307652、rs16671、rs33948716、rs8190507、rs1610937、
rs2307689、rs2307976、rs1305056、rs2067353、rs1610963、rs2307632、rs17878444、
rs140847、rs2307554、rs2308020、rs1610902、rs2307839、rs2307708、rs2308115、
rs3047269,rs2067294,rs2307553,rs16438,rs2307981,rs4646006,rs2308072,rs140864；
For the amplimer by forming with the one-to-one 31 pairs of amplimers of 31 locus, the amplimer is sequence
The nucleotide sequence of SEQ ID No.1 to SEQ ID No.62 in list.
8. a kind of detection kit, which is characterized in that including compound detection system as claimed in claim 7.
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| CN107400713B (en) * | 2017-08-18 | 2020-06-30 | 公安部物证鉴定中心 | Method and system for identifying individuals of Tibetan nationality of Tibet plateau in 27 groups |
| CN108060240B (en) * | 2018-02-12 | 2019-06-04 | 江苏苏博生物医学股份有限公司 | A kind of fluorescence labeling composite amplification kit and its application for insertion deletion detection |
| CN108531572A (en) * | 2018-03-08 | 2018-09-14 | 北京爱普益医学检验中心有限公司 | It is a kind of it is antenatal detection progeny genotypes method and application |
| CN112011622B (en) * | 2019-05-29 | 2022-12-02 | 公安部物证鉴定中心 | Method and system for analyzing non-east Asia and European population sources of individuals with unknown sources |
| CN113322329B (en) * | 2021-05-14 | 2023-03-14 | 公安部物证鉴定中心 | DIP rapid amplification detection reagent for fully integrated microfluidic chip and application thereof |
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